Method for removing osmium from nucleosides

ABSTRACT

A process for removing osmium from an aqueous solution of nucleosides comprising adding hydrogen sulfide, an aromatic pi base such as pyridine, and a mineral acid. The osmium is precipitated out of solution.

This is a division of U.S. Ser. No. 07/772,738 filed Oct. 7, 1991 whichissued at U.S. Pat. No. 5,233,041 on Aug. 3, 1993.

BACKGROUND OF THE INVENTION

Carbocyclic analogues of nucleosides are described in European PatentApplication Publication No. 345,076 published Dec. 6, 1989 as beinguseful as pharmaceuticals in the treatment of viruses, especiallyHerpetoviridae. A particular compound described is (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-hydroxymethyl-1-cyclopentyl]-6H-purin-6-oneof the following formula (I): ##STR1##

European Patent Application Publication No. 349,242 published Jan. 3,1990 assigned to the Wellcome Foundation teaches 6-substituted purinecarbocyclic nucleosides and Example 45 provides a synthesis of(±)-9-[3-(hydroxymethyl)-3-cyclopenten-1-yl)guanine (racemic compound offormula V)).

SUMMARY OF THE INVENTION

Synthetic steps and intermediates involved in the overall reactionscheme of converting carbovir, e.g. (-)-carbovir described in U.K.Patent Application Publication No. 2,217,320A, or (±)-carbovir or(±)-carbovir, of the following formula (II) to the triol of thefollowing formula (I): ##STR2## The individual steps include protectionof the amino group of (II), oxidation of the alcohol to an aldehyde,migration of the cyclopentene double bond to be in conjugation with thealdehyde, reduction back to an alcohol and cis-hydroxylation of thedouble bond.

DETAILED DESCRIPTION OF THE INVENTION

The present invention includes the method of synthesizing a triol of thefollowing formula (Ip): ##STR3## wherein Pro is an amino protectinggroup, which comprises the steps of: i) oxidizing a cyclopentene of thefollowing formula (IIp): ##STR4## to yield an aldehyde of the followingformula (IVp): ##STR5## ii) reducing the aldehyde of formula (IVp) toyield the monoalcohol of the following formula (Vp): ##STR6## and iii)performing a cis-hydroxylation on the monoalcohol of formula (Vp) toproduce the triol of formula (Ip).

Reaction Scheme I which follows sets forth the overall sequence of stepswhich may be used in carrying out the invention. The compound of formula(II) in the following Reaction Scheme I is reacted with an amineprotecting reagent whereby the amino function cannot react withreactants in the subsequent steps, e.g. with (COCl)₂. Thus, Pro informulae (Ip), (IIp), (IIIp), (IVp) and (Vp) represents a moiety boundby a double bond, or 2 moieties bound by 2 single bonds, to the pendantamino nitrogen, e.g. =CN(CH₃)₂ or --H and --CHO). Although various amineprotecting groups can be used, use of an aminal, also known as anamidine or formamide, has the added benefit of rendering the moleculemore soluble in organic solvents so as to allow the subsequent reactionsto proceed more readily. To produce an aminal, the compound of formula(11) is reacted with a dialkyl acetal of a formamide of formula Alk₂NCHO. Acetal reactants thus include those of the formula Alk₂ NCH(OAlk)₂wherein Alk is independently alkyl, in particular of about 1 to 6carbons. Other amine protection includes urethanes which can be producedby reaction of the amine with a chloroformate e.g. of formula ClCOOCH₂CH₃. Reaction with the amine protecting group reagent can be at about25° to 100° C. in an organic solvent such as methanol, benzene ordimethylformamide. ##STR7## Removal of the amine protecting group can becarried out by techniques including acid- or base-catalyzed hydrolysesor hydride reduction. In more detail the hydrolyses can be carried outwith acids such as hydrochloric acid and sulfuric acid and the like orbases such as potassium hydroxide, sodium hydroxide, sodium methoxideand the like or pyridine and triethylamine and the like in proticsolvents such as water, methanol, ethanol and the like. The amineprotecting group (Pro=CNMe₂) can also be removed by hydride reductionwith metal hydrides such as sodium borohydride and the like in proticsolvents such as water, methanol, ethanol and the like. Under especiallymild hydride reduction conditions, (Vp) with the amine protecting groupPro=CNMe₂ can be convened to (Vp) with the amine protecting group Pro=H,CHO as well as (V).

From formula (IIp), the compound of formula (IVp) is then produced by anoxidation, in particular via a Swern oxidation through the unstableintermediate (IIIp). When the alcohol function of (IIp) becomes analdehyde, the double bond in (IIIp) spontaneously migrates to be inconjugation with the carbonyl of the aldehyde. Conditions for a Swernoxidation are generally excess molar equivalent amounts of oxalylchloride, DMSO and triethylamine and a temperature of about -78° to +25°C. A review of the Swern oxidation is found in the article by A. J.Mancuso and Daniel Swern in Synthesis, pp 165-185, March 1981.

The compound of formula (IVp) is then either reduced to thecorresponding alcohol (Vp) or reduced and deprotected simultaneously toyield (V). In the reduction to (Vp), an alkal metal borohydride, such asNaBH₄ NaBH₃ CN may be used in an alcohol such as methanol at about -10°to 0° C., e.g. with 0.25 molar equivalents of NaBH₄ or 1.5 molarequivalents of NaBH₃ CN. If the reduced and deprotected product (V) isdesired directly, the reduction may be carried out with NaBH₄ in ethanolor a higher alcohol in an amount of at least 1 equivalent at about 0° to25° C.

The compound of formula (Vp) and the compound of formula (V) may then bespecifically 1,2-cis-hydroxylated with a catalyst such as osmiumtetroxide (OsO₄) and an oxygen source. Reviews of osmium tetroxideoxidations include those of Martin Schroder in Chemical Reviews, 1980,80, pp 187-213 and V. VanRheenen in Tetrahedron Letters, No. 23 pp.1973-1976. In general, the reaction of (Vp) to (Ip) or (V) to (I) may beconducted in an H₂ O:acetone mixture having a ratio of 1:1 to 50:1, atabout 0° to 100° C., e.g. room temperature, with about 0.008 to 1equivalent of osmium tetroxide and an oxygen source such as hydrogenperoxide, N-methylmorpholine N-oxide, a metal chlorate, t-butylhydroperoxide, sodium peroidate, oxygen gas or sodium hypochlorite.

Also part of the present invention are novel intermediates, e.g. offormulae (Ip), (IIp), (IIIp), (IVp), (Vp) and all enantiomers anddiastereomers thereof and the (1'R)-and (1'S)-enantiomers of (V).

Further parts of the present invention include triols of formula (I) inthe form of the i) hydrochloride, ii) hydrochloride monohydrate, andiii) hemihydrochloride monohydrate. The hydrochloride monohydrate isparticularly important since it has a relatively lower melting pointthan other salts, is not hydroscopic and becomes increasingly purethrough repeated recrystallizations and is thus suitable for use as anactive ingredient in a formulated pharmaceutical. Another aspect of theinvention is a method of removing osmium contamination from a nucleosidewhich comprises:

a) dissolving said nucleoside in a solvent to form a solution,

b) contacting said solution with a precipitating agent,

c) precipitating an osmium-containing residue from the solution, and

d) removing said residue from said solution.

In particular, the precipitating agent may be hydrogen sulfide, anaromatic pi base such as a pyridine, e.g. pyridine itself or a methylmono-, di- or tri-substituted pyridine, or a mineral acid such ashydrochloric acid. Particular combinations of these precipitating agentsare effective such as hydrogen sulfide, pyridine and water followed byhydrogen sulfide, hydrochloric acid and water over a period of up to 7days.

In the following Examples and throughout the specification, thefollowing abbreviations may be used: mg (milligrams); g (grams); ml(milliliters); hr (hours); min (minutes); mp (melting point); mmole(millimoles); MeOH (methanol); ICP-AA (Inductively Coupled Plasma-AtomicAbsorption); and DMSO (dimethyl sulfoxide). Unless otherwise noted, alltemperatures are expressed in ° C. (degrees centigrade).

EXAMPLE 1 a.(1'R-cis)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-6H-purin-6-oneDimethylaminal (Formula (IIp)) purin-6-one Dimethylaminal (Formula(IIp))

A 250 ml round-bottomed flask equipped with a condenser connected to agas inlet was charged with 11.61 g (0.047 mole) of(1'R-cis)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-6H-purin-6-one of formula (II) and 110 ml of methanol under anitrogen atmosphere. The mixture was heated to 65° C. and 9.8 ml (8.39g, 0.070 mole) of dimethylformamide dimethylacetal was added. Themixture was heated to reflux and an additional 3.2 ml (2.74 g, 0.023mole) of dimethylformamide dimethylacetal was added. The mixture washeated for 20 min at reflux, cooled to room temperature, andconcentrated under vacuum to a 30 ml volume. A total of 110 ml ofdiethyl ether was added and the crystalline slurry was stirred briefly.The mixture was filtered and the crystalline solid was washed with 30 mlof diethyl ether. The product was dried under vacuum to constant weightto yield 13.27 g of(1'R-cis)-2-amino-1,9-dihydro-9-[4-hydroxymethyl)-2-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal as a white solid, mp 211°-213° C.; [α]_(D) ²² -170° (c0.21, MeOH).

b.(1'S)-2-Amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1yl]-6H-purin-6-oneDimethylaminal. (Formula (IVp))

A 5 ml flask fitted with a gas inlet and a rubber septum was chargedwith 0.050 g (0.0004 mole) of oxalyl chloride and 1.0 ml of methylenechloride and the solution was cooled to -48° C. under a nitrogenatmosphere. A solution of 0.056 ml (0.062 g, 0.0008 mole) of dimethylsulfoxide in 0.050 ml of methylene chloride was added to the mixture viaa syringe and the mixture was stirred at -48° C. for 5 minutes. Asolution of 0.10 g (0.0003 mole) of(1'R-cis)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal, the product of Example 1a., in 0.60 ml of a 1:1 mixtureof dimethyl sulfoxide-methylene chloride was added via syringe and thismixture was stirred at -48° C. for 10 minutes. The mixture was thenallowed to warm to -18° C. and 0.275 ml (0.0020 mole) of triethylaminewas added via syringe. The mixture was stirred at -18° C. for 10 minutesthen allowed to warm to 0° C. and stirred for 1 hr. During this time,the initially formed(1'R-cis)-2-amino-1,9-dihydro-9-[4-formyl-2-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (IIIp) underwent base-catalyzed double bondmigration. The mixture was poured into 25 ml of saturated aqueous sodiumbicarbonate solution and this mixture was extracted three times with 25ml portions of methylene chloride. The combined organic layers weredried over 1.0 g of anhydrous magnesium sulfate, filtered, andconcentrated under vacuum to yield 0.148 g of an oil. Purification ofthe oil by flash column chromatography on 40-63 μm silica gel andeluting with 9:1 chloroform:methanol provided 0.082 g of (1'S)-2-amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1-yl]-6H-purin-onedimethylaminal as a foam, [α]_(D) ²² +26° (c 0.41, CHCl₃).

c.(1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]6H-purin-6-oneDimethylaminal (Formula (Vp)) and(1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-one(Formula (V))

In a 25 mi flask was placed 0.223 g (0.00074 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of Example 1b. dissolved in 10 ml of absolute ethanol andthe mixture was cooled to 0° C. A total of 0.028 g (0.00074 mole) ofsodium borohydride dissolved in 2 ml of absolute ethanol was addeddropwise via syringe and the mixture was stirred at 0° C. for 25minutes. The mixture was then quenched with 1.0 ml of water followed bystirring for 30 minutes at room temperature. The reaction mixture wasmixed with 3 g of 230-400 mesh silica gel and concentrated to neardryness under vacuum. The resulting solid was added to a column of 8 gof silica gel. Purification by column chromatography via elution with9:1 chloroform-methanol yielded 0.137 g of the less polar(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal, as a white foam: NMR (200 MHz, CDCl₃) δ9.4 (s, 1H), 7.7(s, 1H), 5.7 (s, 2H), 5.2 (m, 1H), 4.3 (s, 2H), 3.2 (s, 3H), 3.1 (s, 3H)and 3.2-2.4 ppm (m, 4H). The column also yielded 0.048 g of the morepolar(1'R-cis)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneas a white foam: NMR (200 MHz, ⁶ d-DMSO) δ10.5 (s, 1H), 7.6 (s, 1H), 6.5(s, 2H), 5.6 (s, 1H), 4.9 (m, 1H), 4.8 (t, J=6 Hz, 1H), 4.0 (d, J=6 Hz,2H), 2.8 (m, 2H) and 2.5 ppm (m, 2H).

d.(1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6oneFormamide (Formula (Vp)

A 100 mL round-bottomed flask containing 34 mL of deionized water, 0.09mL (0.00112 mole) of 36% aqueous hydrochloric acid and 3.40 g (0.0112mole) of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1yl]-6H-purin-6-onedimethylaminal of example 1 c. was placed in an oil bath preheated to68° C. and stirred at this temperature for 45 min. An additional 0.84 mL(0.0101 mole) of 36% aqueous hydrochloric acid was added to the 68° C.solution, and stirring was continued for an additional 35 min. Thesolution was cooled to 23° C. and the pH was adjusted to pH 7 with 11.2mL of 1N aqueous sodium hydroxide. Crystallization occurred immediately.To complete the crystallization, the slurry was cooled to 0° C. The darkcolored solid was collected by vacuum filtration. The solid was washedwith 150 mL of 1:1 ethanol-acetonitrile, then dried at 23° C. (0.5 mm)for 5 hr. The resulting 2.1 g of tan solid was dissolved in 250 mL of1:1 methanol-deionized water at 65° C. The warm solution was treatedwith 2 g of Darco G-60 carbon, then hot-filtered through a 1/2 inch bedof diatomaceous earth. Crystallization occured in the filtrate duringthe filtration. To complete the crystallization, the filtrate was cooledto 0° C. The solids were collected by vacuum filtration and dried at 23°C. (0.5 mm) for 12 hr yielding 1.8 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneformamide as a white solid contaminated with approximately 20% of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-one,mp 230°-245° C. (decomposed). A 1.12 g sample of this solid wastriturated with 50 mL methanol at 60° C. The solids were collected byvacuum filtration and dried at 23° C. (0.5 mm) for 5 hr to provide 0.80g of pure(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneformamide as a white solid, mp 247°-250° C. (decomposed); [α]_(D) ²²+14.1 (c 0.21, DMF). ¹ H-NMR (200 MHz, ⁶ d-DMSO) δ8.0 (s, 1H), 5.6 (s,1H), 5.1 (m, 1H), 4.9 (bt, 1H), 4.1 (bs, 2H), 2.9 (m, 2H) and 2.6 ppm(m, 2H).

e. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneDimethylaminal (Formula (Ip))

A 500 ml 3-necked round-bottomed flask fitted with a condenser, and agas inlet tube was charged with 2.70 g (0.0089 mole) of(1'S-cis)-2-amino-1,9-dihydro-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (Vp), a product of Example I c., 2.09 g(0.0179 mole) of 97% N-methylmorpholine N-oxide, 115 ml of deionizedwater and 1 ml of acetone. The reaction mixture was placed in an oilbath preheated to 70° C. and aqueous osmium tetroxide (4% w/w, 8.2 ml,1.34 mmol) was added dropwise over 1 min. The mixture was stirred at 70°C. for 2 hr. The mixture was cooled to 0° C. and the mixture wasquenched by adding 0.46 g (0.0044 mole) of sodium bisulfite and stirringfor 20 minutes. The heterogeneous mixture was filtered and the solidswere triturated twice with 30 ml portions of methanol and filtered. Thecombined filtrates were concentrated to dryness and residual water wasremoved under vacuum with an acetonitrile azeotrope. The solids weretriturated twice with 50 ml portions of ether and filtered. Thesefiltrates were evaporated to dryness to yield 4.57 g of solid. Thismaterial was slurried with 40 ml of methanol and 6 g of silica gel andthe heterogeneous mixture was evaporated to dryness. The solid wasplaced on top of a 10 g column of silica gel and the column was elutedwith 97:3→75:25 chloroform:methanol. The desired fractions were combinedand evaporated to dryness to yield 2.25 g of solid. This material wasdissolved in 15 ml of hot water, filtered and cooled to 0° C. to inducecrystallization. Isolation of the product afforded 1.65 of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onedimethylaminal as a white solid, mp 263°-268° C. (decomposed); [α]_(D)²² +77.6 (c 0.14, MeOH).

f. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneanhydrous Hydrochloride (Salt (Formula I))

In a 50 ml flask was placed 1.48 g (0.0044 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onedimethylaminal, the product of Example 1e., the 7.5 ml of deionizedwater and 1.1 ml of concentrated hydrochloric acid and the mixture wasstirred at 50° C. for 45 minutes under a nitrogen atmosphere. The hotsolution was filtered and the filtrate was concentrated to 4 ml undervacuum. A total of 15 ml of absolute ethanol was added and the mixturewas heated until homogeneous with the addition of 5 ml of absoluteethanol. The mixture was slowly cooled to room temperature, stirred for2 hr and an additional 4 ml of absolute ethanol was added to fullyinduce crystallization. The solids were isolated by filtration and driedat 100° C. (0.2 mm) for 12 hours to yield 1.05 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneanhydrous hydrochloride salt as a hydroscopic white solid, mp 229°-230°C. (decomposed); [α]_(D) ²² +14.5° (c 0.11, H₂ O); residual osmium was50 ppm by ICP-AA.

g. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1cyclopentanyl]-6H-purin-6-one(Formula (I))

A mixture of 0.273 g (0.00086 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride, 2 ml of deionized water and 0.86 ml of 1N aqueous sodiumhydroxide was heated to induce homogeneity under a nitrogen atmosphere.The solution was cooled to room temperature to induce crystallizationand the solid was isolated in two crops. The solids were collected byfiltration and were dissolved in 80 ml of hot methanol. The hot solutionwas filtered and concentrated to ca. 10 ml to induce crystallization.The 0.170 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onefree base was obtained in two crops as an off-white solid, mp>280° C.(decomposed); [α]_(D) ²² +13.3° (c 0.17, H₂ O).

h. (1' S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneSodium Salt (Formula (I))

1. A mixture of 0.78 g (0.0025 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride salt, 5 ml of deionized water and, 0.245 g (0.0061 mole)of sodium hydroxide dissolved in 0.5 ml of deionized water was stirredat room temperature for 5 minutes. An additional 5 ml of deionized waterwas added and followed by enough absolute ethanol to produce a cloudysolution. The mixture was heated until homogeneous then cooled to roomtemperature to produce an oily two-phased solution. The mixture wasevaporated to dryness under vacuum using an acetonitrile azeotrope. Theremaining oil was triturated with 80 ml of hot methanol. The residualsolid salts were removed by filtration. The mixture was concentrated to15 ml and acetonitrile was added to the cloud point. The mixture washeated to induce homogeneity, then was cooled to room temperature toinduce crystallization. The solid first crop was collected and thefiltrate was concentrated to half volume and an equal volume of hotethanol was added to induce a second crop of crystals. The combinedcrops were dissolved in hot methanol, concentrated to 15 ml and stirredat room temperature to produce 0.420 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onesodium salt as a white solid, mp>265° C.; [α]_(D) ²² +6.6° (c 0.24, H₂O).

2. A solution of 0.491 g (0.0123 mole) of sodium hydroxide in 250 ml ofmethanol was heated to 64° C. The hot solution was added to a 500 mlround-bottomed flask containing 3.90 g (0.0123 mole) of 98+% pure (1'S,3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-one.The white solids went into solution for a moment, then immediatelyprecipitated out as a white, free flowing powder. The slurry was stirredfor 30 minutes and allowed to cool to room temperature. The solventvolume was reduced to 35 ml in vacuo, and 60 ml of diethyl ether wasadded at once. The slurry was stirred for 30 minutes and the whiteprecipitate was collected by vacuum filtration under a blanket ofnitrogen. The resulting hydroscopic solid was dried in vacuo (0.5 mm,65° C., 24 h) to provide 3.72 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onesodium salt, mp>278° C. (decomposed), [α]_(D) ²² +9.86°(c 0.21, H₂ O);residual osmium was 1.6 ppm by ICP-AA.

EXAMPLE 2 a.(1'S)-2-Amino-1,9dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-6-oneDimethylaminal (Formula (Vp))

The following Example represents a combination of the procedures ofExamples of 1b and 1c with isolation of a single product.

A 1 L four-necked, round-bottomed flask fitted with a mechanicalstirrer, a gas inlet tube, a thermometer, and a pressure-equalizeddropping funnel and a rubber septum was charged with 200 ml of methylenechloride under a nitrogen atmosphere and the solution was cooled to -48°C. A total of 4.0 ml (5.35 g, 0.041 mole) of oxalyl chloride was addeddirectly to the solution by syringe. The addition funnel was chargedwith 11.8 ml of a 1:1 mixture of dimethyl sulfoxide and methylenechloride and this solution was added dropwise to the reaction mixture ata rate such that the temperature remained below -35° C. The solution wasstirred for 5 minutes as the mixture was recooled to -48° C. Theaddition funnel was charged with 10.0 g (0.033 mole) of(1'R-cis)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-2-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (IIp), in 100 ml of 1:1 dimethylsulfoxide:methylene chloride. This solution was added to the reactionmixture over a 15 minute period. The reaction mixture was then stirredat -48° C. for 5 minutes, then 21.0 ml (0.015 mole) of triethylaminedissolved in 21.0 ml of methylene chloride was added dropwise over a 4minute period. The reaction mixture was warmed in an ice-water bath to0° C. and stirred at 0° C. for 2.5 hr. The reaction mixture was thenpoured into 200 ml of saturated aqueous sodium bicarbonate solution andthis mixture was extracted three times with 150 ml portions of methylenechloride. The combined organic extracts were dried over 10.0 g ofanhydrous magnesium sulfate, filtered and concentrated under vacuum to avolume of 30 ml which contained crude(1'S)-2-amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal. This concentrate was diluted with 30 ml of methanol andcooled to 0° C. A solution of 0.63 g (0.017 mole) of sodium borohydridein 15 ml of absolute ethanol was added dropwise over 3 minutes. Themixture was stirred at 0° C. for 25 minutes and excess sodiumborohydride was quenched by the addition of 1.0 ml of water. Thesolution was mixed with 30.0 g of 230-400 mesh silica gel and evaporatedunder vacuum to near dryness. The resulting solid was added to a columnof 80.0 g of 230-400 mesh silica gel. Purification by columnchromatography via elution with 95:5→85:15 chloroform:methanol afforded6.77 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal as a white solid mp 108°-110° C.--solidified and remeltedat 194°-195° C.; [α]_(D) ²² -10.2° (c 0.19, MeOH).

b. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneDimethylaminal (Formula) Ip))

A 500 ml, four-necked round-bottomed flask equipped with an air-drivenstirrer, a reflux condenser and a nitrogen/thermometer inlet was chargedwith 20.0 g (0.0662 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (Vp), 15.5 g (0.128 mole) ofN-methylmorpholine-N-oxide, 240 ml of deionized water and 48 ml ofacetone. The slurry became a homogeneous solution as it was heated to70° C. under a nitrogen atmosphere. To the solution was added 10 ml (1.6mmol) of aqueous osmium tetroxide (4% wt/wt) via syringe. The solutionwas stirred at 70° C. for 1 hour and cooled to 10° C. The dark greensolution was vacuum filtered and the filtrate was concentrated in vacuoto a dark colored semisolid. The dark solid was triturated with 1.0 L ofmethanol at 60° C., then filtered. This trituration process was repeatedtwice. The combined filtrates were concentrated in vacuo to give 30.2 gof a brown solid. Purification by flash chromatography (300 g of 40-63μm silica gel using chloroform/methanol/acetic acid, 9:1:0.1 to 4:1:0.1)provided an oil which solidified upon trituration with 2×250 ml toluene.The resulting solids were dried in vacuo (0.5 mm, 63° C., 12 hr) to give18.9 g, of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onedimethylaminal in 85% yield as a white solid, mp 234°-236° C. (dec);[α]_(D) ²² +68° (c 0.05, methanol).

c. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6oneHydrate (Formula (I))

A 3 liter, four-necked round-bottomed flask equipped with an air drivenstirrer, a thermostat-controlled heating mantle, a reflux condenservented through a bleach trap and a fine fritted gas inlet tube wascharged with 42.7 g (0.127 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onedimethylaminal of Formula (Ip) and 1.14 L of deionized water. The slurrywas heated with stirring to 65° C. to form a homogeneous solution, thenhydrogen sulfide gas was gently bubbled into the solution for 2 hours toprecipitate osmium containing impurities. The gas inlet tube wasremoved, the condenser was connected to a nitrogen atmosphere and thereaction mixture was stirred at 65° C. After 22 hours, 600 ml ofabsolute ethanol was added to the warm reaction mixture and the solutionwas hot-filtered through a 2 inch pad (100 g) of diatomaceous earth in ahot, steam-jacketed filter funnel. The pad was washed with 500 mL of 1:1ethanol-water. The combined filtrates were cooled to 25° C., then to 0°C. over 1.5 hr to induce crystallization. The crystals were collected byvacuum filtration. The filtrate volume was reduced in vacuo to 150 mland a second crop of crystals was collected by vacuum filtration. Thedamp 47.0 g of first and second crops were combined with 1.95 L of 60:40methanol-deionized water in a 3 L four-necked round-bottomed flaskequipped with an air-driven stirrer, a reflux condenser vented to ableach bath, a thermometer, and a fine fritted gas inlet tube. Thesolution was heated to 60° C. and hydrogen sulfide was gently bubbledinto the stirred solution for 30 seconds. The solution was stirred for30 minutes at 60° C., then hot filtered through a 2 inch pad (100 g) ofdiatomaceous earth in a hot, steam-jacketed filter funnel. The pad waswashed with 250 ml of 1:1 ethanol:deionized water. The filtrate volumewas reduced in vacuo to 550 ml, then cooled to 0° C. to inducecrystallization. The crystals were collected by vacuum filtration. Thecrystals were washed with 250 ml of absolute ethanol, then 350 ml of 9:1acetonitrile-ethanol to assist in drying. The crystals were transferredto 500 ml round-bottomed flask and dried in vacuo (0.5 mm, 25° C., 5hours) to provide 32.2 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrate in 90% yield. NMR (200 MHz, ⁶ d-DMSO) δ10.6 (s, 1H), 7.8 (s,1H), 6.4 (s, 2H), 4.9 (m, 1H), 4.8 (m, 2H), 4.3 (s, 1H), 4.2 (t, J=6 Hz,1H), 3.4 (m, 2H) and 2.1 ppm (m, 4H).

d. (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneHydrate (formula(I))

The following Example represents a combination of the procedure ofExample 2b and 2c with isolation of a single product.

A 5 L four-necked, round-bottomed flask equipped with an air drivenstirrer, a reflux condenser vented to a bleach trap, a fine fritted gasinlet tube and thermometer was charged with 2.4 L of deionized water and0.6 L of acetone, then heated to 70° C. To the 70° C. solution was added72.6 g (0.620 mole ) of N-methylmorpholine N-oxide and 75.0 g (0.248mole) of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl-6H-purin-6-onedimethylaminal of formula (Vp) followed by 1.0 g (0.0039 mole) of osmiumtetroxide. The solution was stirred at 70° C. for 35 minutes, and 500 mlof pyridine was added. The solution was saturated with hydrogen sulfidegas and stirred at 70° C. for 96 hr to reduce osmate esters and removecolloidal osmium containing impurities. The solution was resaturatedwith hydrogen sulfide every 24 hr. Activated carbon (20 g, Darco G 60)was added to the reaction mixture to remove precipitated osmiumcontaining impurities and elemental sulfur. The hot solution was vacuumfiltered through filter paper and the filtrate was concentrated in situto a dark colored, viscous slurry of about 75 ml. The semi-solid slurrywas diluted with 700 mL of acetonitrile at 23° C. and this slurry wasstirred for 12 hr. The resulting solids were collected by vacuumfiltration to yield 76 g of damp material. The tan colored solids weredissolved in 2.6 L of deionized water at 80° C. To maintain solubility,700 mL of absolute ethanol was added to the 80° C. solution. This hot,homogeneous solution was decolorized with 20 g of activated carbon(Darco G 60) then hot filtered through a 1 inch bed (100 g) ofdiatomaceous earth in a steam jacketed filter funnel. The filtratevolume was reduced to 1.5 L by vacuum distillation to initiatecrystallization. The slurry was stirred for 12 hr at 23° C., then 2 hrat 0° C. to fully effect crystallization. The solids were collected byvacuum distillation to initiate crystallization. The solids werecollected by vacuum filtration, washed with 200 ml of absolute ethanolthen dried in vacuo (0.5 mm, 23° C., 12 hr) to provide 54.0 g of (1'S,3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrate as a white solid containing 8.4 ppm residual osmium asdetermined by ICP-AA. A 5 L four-necked, round-bottomed flask equippedas previously described was charged with 1.2 L of deionized water, 48.5ml of 36% concentrated hydrochloric acid and 54.0 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihyroxy-3-(hydroxymethyl)-1-cylclopentanyl]-6H-purin-6-one hydrate. The solution was heated to 70° C., saturated withhydrogen sulfide gas and stirred for 24 hr. The hot solution was vacuumfiltered through Whatman P3 filter paper to remove osmium containingimpurities and elemental sulphur. The filtrated was returned to a 5 Lfour-necked, round-bottomed flask equipped as previously described. Thesolution was heated to 80° C. and the solution was adjusted to pH 7 withabout 40 mL of 30% aqueous ammonium hydroxide. To maintain solubility,500 mL of ethanol was added to the 80° C. solution. The hot solution wasdecolorized with 20 g of activated carbon, then hot filtered through a 1inch bed (10 g) of diatomaceous earth in a steam jacketed filter funnel.The filtrated volume was reduced in situ to 900 ml to effectcrystallization. To complete the crystallization process, the slurry wasstirred for 3 hr at 23° C., then 1 hr at 0° C. The solids were collectedby vacuum filtration, washed with 100 mL of water at 0° C., 100 ml of1:1 water/absoute ethanol at 0° C., then 100 ml of absolute ethanol atroom temperature. The white crystals were dried in vacuo (0.5 mm, 65°C., 24 hr) to provide 46.3 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrate in 66% yield, mp>199°-204° C. (decomposed), [α]_(D) ²² +13° (c0.17, H₂ O), residual osmium was 0.8 ppm by ICP-AA.

EXAMPLE 3 (1'S, 3'S, 4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-one(Formula (I))

A 250 ml round-bottomed flask equipped with a magnetic stirrer and areflux condenser vented through a nitrogen atmosphere was charged with1.0 g (0.0040 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3cyclopenten-1-yl]-6H-purin-6-oneof formula (V), 1.18 g (0.0101 mole) of N-methylmorpholine N-oxide, 32ml of deionized water and 8 ml of acetone. The soution was placed in anoil bath preheated to 70° C., and 1.25 ml (0.000202 mole) of aqueousosmium tetroxide (4% wt/wt) was added by syringe over 1 minute. Thecolor of the solution changed from pale yellow to deep amber after theaddition. After stirring 50 minutes at 70° C., 2.1 g (0.0202 mole) ofsodium bisulfite was added. The reaction mixture was stirred for 2 hoursat 70° C., then vacuum filtered through 8 g of diatomaceous earth. Thefiltrate was transferred to a 250 ml round-bottomed flask andconcentrated under vacuum. The resulting solids were triturated with 50ml of 9:1 acetonitrile:ethanol. The liquid phase was decanted and thesolids were dried in vacuo. The solids were then dissolved in 30 ml ofdeionized water at 70° C. and filtered. The filtrate was cooled to 0° C.to induce crystallization and the crystals were collected by vacuumfiltration. The solids were washed with 50 ml of acetonitrile,transferred to a 100 ml round-bottomed flask and dried in vacuo (0.5 mm,25° C., 12 hours) to provide 0.824 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-one,mp>265° C. (decomposed); [α]_(D) ²² +13.7° (c 0.07, MeOH); 336 ppmresidual osmium by ICP-AA.

EXAMPLE 4(1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-one(Formula (V))

A solution of 2.25 g (0.00744 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (Vp), 8.3 ml (0.0083 mole) of 1N aqueoussodium hydroxide and 12 ml of deionized water was placed in 100 mlround-bottomed flask equipped with a magnetic stir bar and a refluxcondenser. The solution was stirred for 30 minutes at 23° C., thenwarmed to 65° C. and stirred for 80 minutes. The solution was cooled to23° C. and the pH was adjusted to pH 7 with 0.5 ml of glacial aceticacid in 10 ml of deionized water to induce crystallization. The slurrywas cooled to 0° C., and the crystals were collected by vacuumfiltration. The damp, white crystals were washed with 10 ml ofacetonitrile, transferred to a 100 ml round-bottomed flask and dried invacuo (0.5 mm, 23° C., 12 hours) to provide 1.43 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-one,mp 260°-265° C. (decomposed); [α]_(D) ²² +12.5° (c 0.12, MeOH).

EXAMPLE 5 (1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneDimethylaminal (Formula (Vp))

A solution of 1.16 g (0.00386 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (IVp) in 25 ml of methanol was placed in a 100ml round-bottomed flask equipped with a magnetic stir bar and a refluxcondenser vented to a nitrogen atmosphere. The solution was cooled to-10° C. and 0.039 g (0.0010 mole) of powdered sodium borohydride wasadded at once. The solution was stirred for 20 minutes at -10° C. thenconcentrated in vacuo onto 1 g of 230-400 mesh silica gel. This solidwas added to the top of a small column of 230-400 mesh silica gel andwas chramotographed on a column of 230-400 mesh silica gel using 9:1chloroform:methanol as eluant, provided 0.86 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal as a white foam: NMR (200 MHz, ⁶ d-DMSO) δ11.3 (s, 1H),8.6 (s, 1H), 7.8 (s, 1H) 5.6 (s, 1H), 5.2 (m, 1H), 4.9 (t, J=6Hz, 1H),4.1 (d, J=6Hz, 2H), 3.1 (s, 3H), 3.0 (s, 3H), 2.8 (m, 2H) and 2.5 ppm(m, 2H).

EXAMPLE 6(1'S)-2-Amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneHydrate (Formula (V))

A solution of 0.400 g (0.00133 mole) of(1'S)-2-amino-1,9-dihydro-9-[4-formyl-3-cyclopenten-1-yl]-6H-purin-6-onedimethylaminal of formula (IVp) in 15 ml of absolute ethanol was placedin a 100 ml round-bottomed flask equipped with a magnetic stir bar and areflux condenser vented to a nitrogen atmosphere. The solution wascooled to 0° C. and 0.051 g (0.00133 mole) of powdered sodiumborohydride was added at once. The solution was warmed to 23° C. andstirred for 3.5 hours. To the solution was added 0.11 ml (0.00133 mole)of 36% aqueous hydrochloric acid. The reaction was stirred for 30minutes at 23° C., then concentrated in vacuo onto 2 g of 230-400 meshsilica gel. Purification by flash chromatography (4 g of 230-400 meshsilica gel, eluted with 4:1 chloroform:methanol) provided 0.32 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-oneas a glass-like solid. The solid was dissolved in 10 ml of hot ethanoland the volume was reduced to 4 ml in vacuo when crystals formed. Tothis mixture was added 25 ml of acetonitrile. The resulting slurry wascooled to 0° C., and the crystals were collected by vacuum filtrationthen dried in vacuo (0.5 mm, 23° C., 5 hours) to provide 0.24 g of(1'S)-2-amino-1,9-dihydro-9-[4-(hydroxymethyl)-3-cyclopenten-1-yl]-6H-purin-6-onehydrate as a white solid, mp 148°-160° C. (decomposed); [α]_(D) ²² +14°(c 0.20, MeOH).

EXAMPLE 7 (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneHydrochloride Monohydrate Salt (Formula (I))

A hydrochloric acid solution was prepared by charging an Erlenmeyerflask with 230 ml (2.76 moles) of concentrated hydrochloric acid(Baxter, Baker analyzed) and 645 ml of deionized water. The solution wasstirred and cooled to 20°-25° C. A 5 L four-necked round-bottomed flaskequipped with a glass rod air stirrer, a thermometer, a thermometerinlet, a condenser and a powder funnel was charged with 625 mL of thehydrochloric acid solution and 258.3 g (0.918 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-(3,4-dihydroxy-3-hydroxymethyll-1-cyclopentyl)-6H-purin-6-oneof formula (I) was added. Another 50 ml of the hydrochloric acidsolution was used to complete transfer. Complete solution occurred in5-10 minutes following the addition. The solution was filtered throughWhatman P4 filter paper to remove any insoluble particulate matter. Theresidual material was washed with 100 ml of the hydrochloric acidsolution. The wash and filtrate were combined and returned to a clean 5L four-necked round-bottomed flask equipped as previously described.Another 100 ml of the hydrochloric acid solution was used to completethe transfer. Then the solution was stirred at 20°-25° C. while adding3.5 L of absolute ethanol over a 30-minute period to inducecrystallization. Following completion of the alcohol addition, the whiteslurry was stirred for 30 minutes at 20°-25° C. Then the slurry wascooled to 0°-5° C. over a 30-minute period then was stirred at 0°-5° C.for 1 hr before isolating the product by filtration on Whatman P4 filterpaper. The solids were washed with 2×125 ml=250 ml of absolute ethanolat 0°-5° C. The product was placed in a vacuum oven at 63° C. (1 mm) todry to constant weight, providing 263.9 g of white crystalline (1'S,3'S, 4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride monohydrate salt.

A 2 L Erlenmeyer flask equipped with magnetic stir bar was charged with138.4 ml (1.66 moles) of 36% concentrated hydrochloric acid and 553 mlof deionized water. A 250 ml aliquot of this hydrochloric acid solutonwas retained for rinses. The hydrochloric acid solution was heated to45° C. before adding 263.0 g (0.783 mole) (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride monohydrate salt of formula (I). The material was rinsedin with 250 ml of hydrochloric acid solution to complete the transfer.Complete solution occurred in 5-10 minutes following the addition. Thesolution was heated to 65° C. and filtered through Whatman P4 filterpaper to remove any particulate matter. The residual material was washed2×50 ml=100 ml of deionized water. The wash and filtrate were combinedand transferred to a 5 L four-necked round-bottomed flask equipped witha glass rod air stirrer, a thermometer inlet, a thermometer, a condenserand an addition funnel. The transfer was completed using 38 mL ofdeionizd water. The solution was stirred at 50° C. while adding 3.5 Labsolute ethanol over a 30-minute period to induce crystallization. Thethick white slurry was stirred for 1 hr before isolating the product byfiltration on Whatman P4 paper. The product was washed with 2×250 ml=500ml of absolute ethanol at 0°-5° C. The product was dried in a vacuumoven at 63° C. (1 mm) to constant weight to provide 232.0 g of (1'S,3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride monohydrate salt as a white solid, mp 172°-179° C.(decomposed); [α]_(D) ²² +15.8° (c 0.50, H₂ O).

EXAMPLE 8 (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneHemihydrochloride Monohydrate Salt (Formula (I))

A 2 L Erlenmeyer flask equipped with a magnetic stir bar was chargedwith 27 ml of deionized water and 9.0 g (0.027 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride monohydrate salt of formula (I). The slurry was heated to65° C. and complete solution occured at 45° C. The hot solution wasfiltered through Whatman P4 filter paper to remove any insolubleparticulate matter. Another 9 ml of deionized water was used to completethe transfer and wash the residue. The combined wash and filtrate weretransferred to a 500 ml four-necked round-bottomed flask equipped with aglass rod air stirrer, a thermometer inlet, a thermometer, a condenserand an addition funnel. The hot solution was stirred and 130 ml ofabsolute ethanol was added over a 30-minute period. The slurry wasstirred for 2 h while cooling to 20°-25° C., then was cooled withstirring to 0°-5° C. over a 30-minute period. The slurry was stirred foran additional 2 hr at 0°-5° C. before isolating the product byfiltration. The solids were washed with 2×15 ml=30 ml of absoluteethanol at 0°-5° C. The product was dried in a vacuum oven at 63° C. toconstant weight providing 7.0 g of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehemihydrochloride monohydrate salt, mp 241°-243° C. (decomposed);[α]_(D) ²² +14.7° (c 0.41, H₂ O).

EXAMPLE 9 (1'S, 3'S,4'S)-2-Amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneAnhydrous Hydrochloride Salt (Formula (I))

A total of 1.24 g (0.0037 mole) of (1'S, 3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-onehydrochloride monohydrate salt of formula (I) was placed in anevaporating dish and dried in a vacuum oven (103° C., 0.5 mm) for 12 hr.The 1.16 g of the resulting hydroscopic solid was identified as (1'S,3'S,4'S)-2-amino-1,9-dihydro-9-[3,4-dihydroxy-3-(hydroxymethyl)-1-cyclopentanyl]-6H-purin-6-oneanhydrous hydrochloride salt, mp 229°-230° C. (decomposed); [α]_(D) ²²+16.2° (c 0.28, H₂ O).

What is claimed is:
 1. A method of removing osmium contamination from a nucleoside which comprises:a) dissolving said nucleoside in an aqueous solution, b) contacting said solution with hydrogen sulfide, an aromatic pi base and a mineral acid, c) precipitating an osmium-containing residue from the solution, and d) removing said residue from said solution.
 2. The method of claim 1, wherein said aromatic pi base is pyridine. 